General Considerations

Three types of culture system will be discussed in this manual for the cultivation of Entamoeba species: xenic cultivation, where the parasite is grown in the presence of an undefined flora; monoxenic, where the parasite is grown in the presence of a single species of associate; and axenic, where the parasite is grown in the absence of any other metabolizing cells. (The term polyxenic is sometimes erroneously used as a synonym for xenic; however polyxenic should refer only to cultures where the identities of all the species present are known (14)) These parasitic protista are all isolated from sources rich in bacteria and fungi. Controlling or eliminating the latters' growth is crucial to success in cultivating the parasites of interest. This is true even in xenic culture, where a balance between the needs of the bacterial flora and the eukaryote is important.

Unlike Trichomonas vaginalis and Giardia intestinalis (also called G. lamblia and G. duodenalis by some authors) which can readily be established directly into axenic cultures, Entamoeba histolytica has never been grown axenically without first being established in culture with other organisms and usually with a complex undefined bacterial flora. Axenization of E. histolytica is a laborious and lengthy process where xenic cultures of the organism are first adapted to monoxenic growth, usually with a kinetoplastid flagellate as an associate, before weaning them from a phagocytotic lifestyle to one where the nutrients are obtained largely by pinocytosis (9). The whole process often takes months.

One constant problem facing those who rely on axenic cultures of these organisms is their fastidiousness. Lot to lot variations in several components of the axenic culture media in particular can have profound effects on the ability of a medium to support growth of the organisms; some lots may even be toxic. Trypticase (Casein Digest Peptone), yeast extract and serum are the medium components most commonly affected, but the quality of the distilled water and even the type of glass used in making the culture tubes (disposable borosilicate glass should be used) can cause problems (7). For this reason we highly recommend that those wishing to undertake axenic cultivation of these organisms test the ability of each new lot of reagent to support growth before starting to use it.

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